Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Nitzsche, Anja; Pietilä, Riikka; Love, Dominic; Testini, Chiara; Ninchoji, Takeshi; Smith, Ross O; Ekvärn, Elisabet; Larsson, Jimmy; Roche, Francis P.; Egaña, Isabel; Jauhiainen, Suvi; Berger, Philipp; Claesson‐Welsh, Lena; Hellström, Mats

doi:10.15252/embr.202050218

Cited by 10 publications

(13 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PALD1 contains two protein tyrosine phosphatase (PTP) domains with four minimal active site motifs (CX 5 R) and belongs to the PTP superfamily ( Chen et al, 2017 ). While its exact substrate specificity remains to be firmly established, phosphoinositide phosphatase activity has recently been detected in PALD1 immunoprecipitates ( Nitzsche et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

Time-resolved proteomics profiling of the ciliary Hedgehog response

May

Kalocsay

D’Auriac

et al. 2021

Journal of Cell Biology

View full text Add to dashboard Cite

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

show abstract

Section: Resultsmentioning

confidence: 99%

Time-resolved proteomics profiling of the ciliary Hedgehog response

May

Kalocsay

D’Auriac

et al. 2021

Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…As a component of internalized VEGFR2 is recycled back to the cell surface (Simons et al, 2016), the reduction of cell-surface VEGFR2 may be the result of increased internalization or less recycling. Thus, we used another biotinylation assay (Nitzsche et al, 2021) to quantify internalized VEGFR2 ( Fig. 3C, D ).…”

Section: Resultsmentioning

confidence: 99%

“…For assessment of the internalized pool of VEGFR2 after VEGF stimulation, biotinylation of cell surface proteins was performed before VEGF-A stimulation as described (Nitzsche et al, 2021). Briefly, after serum starvation for 2 hours, cells were incubated 0.2 mg/mL of sulfo-NHS-SS-biotin for 20 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…After biotin labeling on the surface proteins, cells were stimulated with VEGF-A (50 ng/mL) for indicated time points. Cells were washed with cold PBS, and cell surface biotin was cleaved off by incubating the cells on ice with 100 mM of membrane-impermeable reducing agent MESMA (2-mercaptoethane sulphonic acid (Sigma) as described (Nitzsche et al, 2021). Cells were lysed with lysis buffer (10 mM HEPES, 150 mM NaCl, 1% TritonX100, 0.5% NP40, 1 mM EGTA, 1 mM EDTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM beta-glycerophosphate, and protease inhibitor cocktail).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A kinesin mediates VEGFR2 recycling and regulates VE-cadherin phosphorylation, essential for vascular permeability

Cho

Zhou

et al. 2022

Preprint

View full text Add to dashboard Cite

Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A in inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and endothelial permeability is unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates VE-cadherin phosphorylation and endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle, and inhibition of this recycling attenuated phosphorylation of VEGFR2 at Y951, Src, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, suggesting a promising strategy for mitigating vascular leakage associated with inflammatory diseases.

show abstract

“…Paladin contains two phospho-tyrosine phosphatase consensus active site motifs but no other known phosphatase characteristics. A recent report describes paladin as a phosphatidylinositol 4,5-bisphosphate phosphatase that lacks phospho-tyrosine/serine/threonine protein phosphatase activity [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

Paladin, overexpressed in colon cancer, is required for actin polymerisation and liver metastasis dissemination

et al. 2022

View full text Add to dashboard Cite

Introduction Colorectal cancer remains a public health issue and most colon cancer patients succumb to the development of metastases. Using a specific protocol of pressure-assisted interstitial fluid extrusion to recover soluble biomarkers, we identified paladin as a potential colon cancer liver metastases biomarker. Methods Using shRNA gene knockdown, we explored the biological function of paladin in colon cancer cells and investigated the phospho-proteome within colon cancer cells. We successively applied in vitro migration assays, in vivo metastasis models and co-immunoprecipitation experiments. Results We discovered that paladin is required for colon cancer cell migration and metastasis, and that paladin depletion altered the phospho-proteome within colon cancer cells. Data are available via ProteomeXchange with identifier PXD030803. Thanks to immunoprecipitation experiments, we demonstrated that paladin, was interacting with SSH1, a phosphatase involved in colon cancer metastasis. Finally, we showed that paladin depletion in cancer cells results in a less dynamic actin cytoskeleton. Conclusions Paladin is an undervalued protein in oncology. This study highlights for the first time that, paladin is participating in actin cytoskeleton remodelling and is required for efficient cancer cell migration.

show abstract

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Cited by 10 publications

References 38 publications

Time-resolved proteomics profiling of the ciliary Hedgehog response

Time-resolved proteomics profiling of the ciliary Hedgehog response

A kinesin mediates VEGFR2 recycling and regulates VE-cadherin phosphorylation, essential for vascular permeability

Paladin, overexpressed in colon cancer, is required for actin polymerisation and liver metastasis dissemination

Contact Info

Product

Resources

About